The spinal cord coordinates the body's movement and sensation to and from the brain. It is a complex organ containing nerve cells (also known as neurons), and supporting cells (also known as neuroglial cells or glial cells). A typical neuron consists of a cell body, containing the nucleus and the surrounding cytoplasm (perikaryon); several short radiating processes (dendrites); and one long process (the axon, which terminates in twiglike branches (telodendrons) and may have branches (collaterals) projecting along its course. The axon together with its covering or sheath forms the nerve fiber. The neuroglial or glial cells form the supporting structure of nervous tissue. There are three types of glial cells, which are astrocytes, oligodendrocytes, and microglia. Astrocytes and oligodendrocytes (collectively macroglia) are of ectodermal origin. These cells far outnumber neurons in the brain and spinal cord and perform many essential functions. The oligodendrocyte creates the myelin sheaths that insulate axons and improve the speed and reliability of nerve signal transmission. Astrocytes, large star-shaped glial cells, regulate the composition of the fluids that surround neurons. Some of these cells also form scar tissue after injury. Microglia are of mesodermal origin. They are smaller cells that become activated in response to injury and help clean up waste products. All of these glial cells produce substances that support neuron survival and influence axon growth.
There are several types of neurons that lie within the spinal cord and carry out the spinal cord functions. Large motor neurons have long axons that control skeletal muscles in the neck, torso, and limbs. Sensory neurons (also called dorsal root ganglion cells) have axons which carry information from the body into the spinal cord. Spinal interneurons, which lie completely within the spinal cord, help integrate sensory information and generate coordinated signals that control muscles.
Motor neurons are traditionally classified as upper motor neurons or lower motor neurons. Upper motor neurons reside in the precentral gyrus of the brain, and send long processes down to synapse on lower motor neurons in the ventral (anterior) horns of the grey matter of the spinal cord. From the ventral horns of the spinal cord, axon processes of lower motor neurons coalesce to form the ventral roots. These axons eventually terminate on one or more muscle fibers. Through arborization of the terminal part of its fiber, each lower motor neuron comes in contact with anywhere from a few to 100-200 or more muscle fibers to form a “motor unit,” (Adams and Victor, 1985, in “Principles of Neurology,” McGraw-Hill, Inc., New York, p. 37).
Many axons in the spinal cord are covered by sheaths of an insulating substance called myelin, which gives them a whitish appearance; therefore, the region in which they lie is called “white matter.” The neurons themselves, with their tree-like dendrites that receive signals from other neurons, make up “gray matter.” This gray matter lies in a butterfly-shaped region in the center of the spinal cord. Like the brain, the spinal cord is enclosed in three membranes (meninges): the pia mater, the arachnoid, and the dura mater. The spinal cord is then surrounded by rings of bone called vertebra.
The spinal cord and the brain together make up the central nervous system (CNS). Unlike neurons of the peripheral nervous system (PNS), which carry signals to the limbs, torso, and other parts of the body, it is generally believe that neurons of the CNS do not regenerate after injury. (Walker, New Eng. J. Med., (1991) 324: 1885-1887). Recent research, however, supports that the injured spinal cord can be successfully regenerated, provided that the damaged neurons must be able to survive the injury or be replaced, and the axons must be able to regrow and find appropriate targets. Axons and their targets must then interact to construct synapses, the specialized structures that act as the functional connections between neurons.
The spinal cord is about 18 inches long and extends from the base of the brain, down the middle of the back, to about the waist. The spinal cord is organized into segments along its length. The spinal nerves that branch out from the spinal cord to the other parts of the body are lower motor neurons (LMNs). Thirty-one pairs of spinal nerves exit and enter at each vertebral level and communicate with specific areas of the body. The segments in the neck, or cervical region, referred to as C1 through C8, control signals to the neck, arms, and hands. Those in the thoracic or upper back region (T1 through T12) relay signals to the torso and some parts of the arms. Those in the upper lumbar or mid-back region just below the ribs (L1 through L5) control signals to the hips and legs. Finally, the sacral segments (S1 through S5) lie just below the lumbar segments in the mid-back and control signals to the groin, toes, and some parts of the legs. The effects of spinal cord injury at different segments reflect this organization.
Neurons of the brain and spinal cord respond to signals differently from most other cells of the body, including those in the peripheral nervous system (PNS). The brain and spinal cord (i.e., the CNS) are confined within bony cavities that protect them, but also render them vulnerable to compression damage caused by swelling or forceful injury. Cells of the CNS have a very high rate of metabolism and rely upon blood glucose for energy. The “safety factor,” that is the extent to which normal blood flow exceeds the minimum required for healthy functioning, is much smaller in the CNS than in other tissues. For these reasons, CNS cells are particularly vulnerable to reductions in blood flow (ischemia). Other unique features of the CNS are the “blood-brain-barrier” and the “blood-spinal-cord barrier.” These barriers, formed by cells lining blood vessels in the CNS, protect neurons by restricting entry of potentially harmful substances and cells of the immune system. Trauma may compromise these barriers, perhaps contributing to further damage in the brain and spinal cord. The blood-spinal-cord barrier also prevents entry of some potentially therapeutic drugs. Finally, in the brain and spinal cord, the glial cells and the extracellular matrix (the material that surrounds cells) differ from those in peripheral nerves. Each of these differences between the PNS and CNS contributes to their different responses to injury.
Although spinal cord is well protected, spinal cord injury does occur due to causes such as trauma (e.g., car accident, violence, falls, sports) and disease (e.g., polio, spina bifida, Friedreich's Ataxia). In the United States, approximately 450,000 people live with spinal cord injury, and there are about 10,000 new cases every year, mostly involving males between ages of 16 to 30. Spinal cord injury can be divided into complete injury (no motor and sensory functions below the level of the injury) and incomplete injury (some functioning below the primary level of injury). Recovery from spinal cord injury is much more difficult because the neurons are very specialized and unable to divide and create new cells.
Usually, injuries to the spinal cord do not result in a cut through the cord; instead, they crush the axons. Researchers studying spinal cords obtained from autopsy have identified several different types of spinal cord injuries. The most common types of spinal cord injuries are contusions (bruising of the spinal cord) and compression injuries (caused by pressure on the spinal cord). Other types of injury included lacerations, caused by a bullet or other object, and central cord syndrome.
The damage that occurs to spinal cord axons within the first few hours after-injury is complex and it occurs in stages. The normal blood flow is disrupted, which causes oxygen deprivation to some of the tissues of the spinal cord. Bleeding into the injured area leads to swelling, which can further compress and damage spinal cord axons. The chemical environment becomes destructive, due primarily to the release of highly reactive molecules, e.g., free radicals. These molecules break up cell membranes, including killing cells that were not injured initially. Macrophages that invade the site of injury to clean up debris may also damage uninjured tissue. Non-neuronal cells, such as astrocytes, may divide too often, forming a scar that impedes the regrowth of the injured nerve cell axons.
The early events that follow a spinal cord injury can lead to other kinds of damage later on. Within weeks or months, cysts may form at the site of injury which fill with cerebrospinal fluid. Typically, scar tissue develops around the cysts, creating permanent cavities that can elongate and further damage neurons. Also, nerve cell axons that were not damaged initially often lose their myelin. Over time, these and other events can contribute to more tissue degeneration and a greater loss of function.
Effective drug therapy for spinal cord injury first became a realty in 1990s, when methylprednisolone, the first drug shown to improve recovery from spinal cord injury, moved from clinical trials to standard use. The NASCIS II (National Acute Spinal cord Injury Study II) trial, a multicenter clinical trial comparing methylprednisolone to placebo and to the drug naloxone, showed that methylprednisolone given within 8 hours after injury significantly improves recovery in humans. Completely paralyzed patients given methylprednisolone recovered an average of about 20% of their lost motor function, compared to 8% recovery of function in untreated patients. Partially paralyzed patients recovered an average of 75% of their function, compared to 59% in people who did not receive the drug. Patients treated with naloxone, or treated with methylprednisolone more than 8 hours after injury, did not improve significantly more than patients given a placebo. (National Institutes of Health, “Spinal Cord Injury: Emerging Concepts,” (1996) www.ninds.nih.gov/health_and _medical/pubs). The results of the clinical trial of methylprednisolone revolutionized thinking that there is a window of opportunity for acute treatment of spinal cord injury.
Today, more drugs are being tested for treatment of spinal cord injury. For example, U.S. Pat. No. 6,291,493 discloses the use of clomethiazole (5-(2-chloroethyl)-4-methylthiazole) for treating pathological conditions caused by compression to the spinal cord, whether traumatically induced or due to compression caused by tumours, haemorrhage, infectious processes or spinal stenosis. U.S. Pat. No. 6,288,069 discloses the use of 9-substituted hypoxanthine derivatives (such as N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide) to stimulate regeneration or survival of a mammalian motor neuron or of a mammalian sensory neuron. U.S. Pat. No. 6,143,714 discloses the use of a hepatocyte growth factor (HGF) to promote the survival, growth and differentiation of motor neurons.
Recently, Cheung et al. (FEBS Lett 2000; 486(3): 291-6) disclose that Ganoderma lucidum extract induces neuronal differentiation in vitro, using a primary neuronal cell system (i.e., PC12 cells). The PC12 cells are derived from rat pheochromocytoma cells which respond to the nerve growth factor (NGF). The Ganoderma ludicum extract described by Cheung et al. is made from the fruit bodies of the Ganoderma lucidum. 
In the invention to be presented in the following sections, a novel use of the sporoderm-broken germination-activated Ganoderma spores (GASP) from Ganoderma lucidium for treatment of spinal cord injury is undertaken. The GASP have previously been disclosed for use in treating patients with cancer, AIDS, hepatitis, diabetes, and cardiovascular diseases, and can prevent or inhibit free radical oxidation and hepatotoxic effects. See U.S. Pat. Nos. 6,316,002 and 6,468,542, which are incorporated herein by reference. Unlike methylprednisolone, which has to be given to patients within 8 hours after injury to be effective, the GASP can be administered to humans and animals in a much later time while still achieving significant, improvement. A further benefit of using the GASP is that they are non-toxic so that higher dosage can be prescribed to the patients.